We are developing an in vitro model of human esophageal carcinogenesis. Using the strategy proven successful for other types of human epithelial cells, a serum-free medium for normal human esophageal epithelial cells has been developed. The pathobiological effects of 12-0-tetradecanoylphorbol-13-acetate, T-2 toxin, cigarette smoke condensate and ethanol have been examined. For comparison studies with normal human esophageal epithelial cells, seven human esophageal carcinoma cell lines have been newly established in LHC. The carcinoma cell lines had fewer numbers of epidermal growth factor receptors than the normal esophageal epithelial cells. The carcinoma cells were aneuploid with frequent structural abnormalities in chromosomes 1, 3, 9 and 11. A homogeneously staining region at llql2 was found in 3 of the carcinoma cell lines. Since "immortalization" appears to be a rate-limiting step in human cell carcinogenesis in vitro, normal esophageal epithelial cells were transfected by strontium phosphate coprecipitation with a plasmid pRSV-SV40 T and a cell line (HET-IA) has been developed that has undergone more than 160 population doublings. HET-IA has an epithelial morphology, stains for cytokeratins and SV40 T antigen by immunofluorescence, and has remained nontumorigenic in athymic nude mice. We plan a multifaceted approach to neoplastically transform normal or SV40 T "immortalized" (HET-lA) human esophageal epithelial cells. The cells will be exposed to chemical carcinogens (e.g., N-methyl-N'-nitrosourea or benzo[a]pyrene diol epoxide), selected for altered growth properties including resistance to inducers of terminal squamous differentiation and examined for tumorigenicity in athymic nude mice.